The serine protease thrombin occupies a central role in hemostasis and thrombosis (Tapparelli et al., Trends in Pharmacological Sciences 14:366-376 (1993); Lefkovits and Topol, Circulation 90(3):1522-1536 (1994); Harker, Blood Coagulation and Fibrinolysis 5 (Suppl 1):S47-S58 (1994)). Activation of the coagulation cascade through either the intrinsic pathway (contact activation) or the extrinsic pathway (activation by exposure of plasma to a non-endothelial surface, damage to vessel walls or tissue factor release) leads to a series of biochemical events that converge on thrombin. Thrombin cleaves fibrinogen ultimately leading to a hemostatic plug (clot formation), potently activates platelets through a unique proteolytic cleavage of the cell surface thrombin receptor (Coughlin, Seminars in Hematology 31(4):270-277 (1994)), and autoamplifies its own production through a feedback mechanism.
As a multifactorial protein, thrombin induces a number of effects on platelets, endothelial cells, smooth muscle cells, leukocytes, the heart, and neurons (Tapparelli et at., Trends in Pharmacological Sciences 14:366-376 (1993); Church and Hoffman, Trends in Cardiovascular Medicine 4(3):140-146 (1993)). Platelet activation leads to shape change and aggregation as well as the synthesis, release and secretion of vasoactive substances and lysosomal enzymes. Endothelial cell activation results in the secretion of stimulatory agents leading to increased vascular permeability and adhesiveness for mononuclear cells, one consequence of which is extravasation of leukocytes at the site of thrombin generation. Thrombin induces fibroblast and smooth muscle cell proliferation suggesting that thrombin plays a key role in lesion development following vascular damage. Enhanced automaticity and prolongation of repolarization have been observed in cardiac myocytes showing sensitivity to thrombin. Normal neuronal development has been shown also to be influenced by thrombin. Thus, inhibitors of thrombin function have therapeutic potential in a host of cardiovascular and non-cardiovascular diseases, including: myocardial infarction; unstable angina; stroke; restenosis; deep vein thrombosis; disseminated intravascular coagulation caused by trauma, sepsis or tumor metastasis; hemodialysis; cardiopulmonary bypass surgery; adult respiratory distress syndrome; endotoxic shock; rheumatoid arthritis; ulcerative colitis; induration; metastasis; hypercoaguability during chemotherapy; Alzheimer's disease; and Down's syndrome.
To date only three classes of compounds (heparins, low-molecular weight heparins, and coumarins, such as warfarin) have been used in anticoagulant therapy. Each class has severe limitations and liabilities (Weitz and Hirsh, Journal of Laboratory Clinical Medicine 122:364-373 (1993); Raj et al., The American Journal of the Medical Sciences 307(2):128 (1994)). All three classes indirectly inhibit thrombin. Heparin and low-molecular weight heparins augment antithrombin III and/or heparin cofactor II inhibition of thrombin, whereas coumarins inhibit vitamin K-dependent post-translational modification. Close monitoring and titration of therapeutic doses is required when employing these agents due to patient variability. Hemorrhagic complications due to bleeding are an encountered side effect. In fact, bleeding remains as the most common side effect of long term oral anticoagulant therapy. Lack of activity in arterial thrombosis in the case of heparin is due to its inability to inhibit clot bound thrombin. Lack of oral activity in the case of heparins and low-molecular weight heparins preclude their use for chronic administration.
Direct thrombin inhibitors of various structural classes have been identified recently (Tapparelli et al., Trends in Pharmacological Sciences 14:366-376 (1993); Claeson, Blood Coagulation and Fibrinolysis 5:411-436 (1994); Lefkovits and Topol, Circulation 90(3):1522-1536 (1994)). Representative compounds that act by inhibiting the active site of thrombin include the .alpha.-chloroketone D-phenylalanyl-L-prolyl-L-arginyl chloromethylketone (PPACK), the boroarginine DUP714, the peptide arginal GYK114766, the cyclic peptides cyclotheonamides A and B, the benzamidine NAPAP, and the arylsulphonylarginine argatroban. The thrombin inhibitory peptides hirudin and hirulogs additionally span through the active and exosite domains of thrombin. The peptide hirugen and single-stranded DNA aptamers inhibit thrombin through exosite occupancy. These classes of antithrombotic agents still suffer from one or more of the following liabilities: (1) poor oral bioavailability due to the peptidic or oligonucleotidic nature of these agents, or high molecular weight or charged nature of the agents; (2) excessive bleeding complications; (3) poor selectivity towards thrombin versus other serine proteases (which may lead to severe and sometimes fatal hypotension and respiratory depression in animal models); (4) liver toxicity; or (5) cost effectiveness.
An alternative approach for inhibiting thrombin function is to inhibit factor Xa. Factor Xa associates with factor Va and calcium on a phospholipid membrane thereby forming a prothrombinase complex. This prothrombinase complex then converts prothrombin to thrombin (Claeson, Blood Coagulation and Fibrinolysis 5:411-436 (1994); Harker, Blood Coagulation and Fibrinolysis 5 (Suppl 1):S47-S58 (1994)). Inhibitors of factor Xa are thought to offer an advantage over agents that directly inhibit thrombin since direct thrombin inhibitors still permit significant new thrombin generation (Lefkovits and Topol, Circulation 90(3):1522-1536 (1994); Harker, Blood Coagulation and Fibrinolysis 5 (Suppl 1):S47-S58 (1994)). Indeed, continuous generation of new thrombin rather than reexposure of preformed clot-bound thrombin is thought to be responsible in part for the phenomenon of reocclusion since markers of thrombin generation have been found to increase during and after thrombolytic treatment for myocardial infarction. Thus, it is now believed that increased thrombin activity associated with thrombolysis is due at least in part to new thrombin generation.
Specific protein factor Xa inhibitors, such as the leech-derived, 119-amino acid protein antistasin and the soft tick derived protein TAP (tick anticoagulant peptide) accelerated clot lysis and prevented reocclusion when given as adjuncts to thrombolysis (Mellott et al., Circulation Research 70:1152-1160 (1992); Sitko et al., Circulation 85:805-815 (1992)). U.S. Pat. No. 5,385,885, issued Jan. 31, 1995, discloses smooth muscle cell proliferation inhibitory activity of both TAP and antistasin. Additionally, TAP and antistasin have been shown to reduce experimental restenosis. These results suggest that factor Xa may play a role in the restenosis process through its effects upon thrombus formation or through its mitogenic potential (Ragosta et al., Circulation 89:1262-1271 (1994)). The peptide ecotin is another selective, reversible, tight-binding inhibitor of factor Xa that exhibits potent anticoagulant activity (Seymour et al., Biochemistry 33:3949-3959 (1994); PCT Published Application WO 94/20535, published Sep. 14, 1994). Ixodidae, argasin, and ancylostomatin are other representative peptidic factor Xa inhibitors isolated from animals that feed on blood (Markwardt, Thrombosis and Hemostasis 72:477-479 (1994)).
Non-peptide diamidino derivatives, such as (+)-(2S)-2-[4-[[(3S)-1-acetimidoyl-3-pyrrolidinyl]oxy]phenyl]-3-[7-amidino -2-naphthyl]propanoic acid hydrochloride pentahydrate (DX-9065a), exhibit anticoagulant activity (Tidwell et al., Thrombosis Research 19:339-349 (1980); Yamazaki et al., Thrombosis and Hemostasis 72:393-395 (1994); Hara et al., Thrombosis and Hemostasis 71:314-319 (1994); Nagahara et al., Journal of Medicinal Chemistry 37:1200-1207 (1994)). Synthetic amidino derivatives of phenylalanine and cycloheptanone have also shown potent factor Xa inhibition (Sturzebecher et al., Thrombosis Research 54:245-252 (1989)).
PCT Published Application WO 94/13693, published Jun. 23, 1994, discloses peptide analogs containing an aldehyde grouping. The application discloses that the analogs have substantial potency and specificity as inhibitors of mammalian factor Xa.
PCT Published Applications WO 93/15756, published Aug. 19, 1993, and WO 94/17817, published Aug. 18, 1994, disclose peptidyl arginine aldehydes that exhibit factor Xa and/or thrombin inhibitory activity.
PCT Published Applications WO 94/20526, published Sep. 15, 1994, discloses peptide derivatives having a C-terminal boronic acid group. The application discloses that these peptide derivatives possess protein-inhibiting activity and are potent thrombin inhibitors.